Steroid hormones are essential for growth, development, and homeostasis of animals, insects, and plants. In plants, one class of polyhydroxylated steroids, called brassinosteroids (BRs), has wide distribution throughout the plant kingdom and unique growth promoting activities. During the past decade, we have analyzed the biosynthesis and cellular functions of BRs, and identified the membrane-localized receptor, BRI1, as well as many of its downstream signaling components. BRI1 is a Toll-like receptor kinase comprised of a large extracellular ligand-binding domain containing 24 leucine-rich repeats, a trans-membrane segment and a cytoplasmic kinase domain. Previous studies indicate that the extracellular domain of BRI1 binds the steroid hormone, which then induces a conformational change in the receptor that in turn leads to auto- phosphorylation of the cytoplasmic kinase domain and the dissociation of the kinase inhibitor protein BKI1. This increases the affinity of BRI1 for its co-receptor, BAK1, a receptor kinase with 5 LRRs. Extensive cross- phosphorylation events between the kinase domains of the receptor and the co-receptor then lead to a fully activated signaling complex. Here, we propose combining structural biology/biochemistry with genetics/cell biology to dissect the molecular mechanism of the receptor. We want to analyze its mode of ligand recognition and activation, its interaction with the co-receptor BAK1, and the regulation of its kinase activity by the novel inhibitor, BKI1. Thus, this proposal has the following specific aims: (1) Determine the detailed mechanism of steroid hormone recognition by the 94 amino acid steroid-binding domain in context of the LRR modules in the extracellular domain of BRI1;(2) Develop a mechanistic model for receptor activation and test this model in vitro and in vivo;(3) Elucidate the molecular mode of action and specificity of BKI1 and 6 related proteins in the negative regulation of BRI1's kinase activity. Our long-term goal is to develop a validated mechanistic model that accurately describes the stimulation and regulation of the BR signaling pathway. BRI1 is the best studied and most understood of any cell surface receptor proposed for steroid hormones in multicellular eukaryotes. The experiments described here will thus inform our mechanistic understanding of a new paradigm for steroid hormone perception and signaling from a cell surface receptor. The strength of Arabidopsis as a genetic model has allowed the identification of dozens of mutant alleles (both gain- and loss-of-function) in both the receptor and its co-receptor, as well as elucidated an entire signaling pathway from the receptor to changes in nuclear gene expression. Thus, we are well-poised to interpret the structural studies. BRI1 is a founding member of the largest family of receptor kinases in plants, and shares significant homology with mammalian innate immunity receptors. As such, our studies will provide mechanistic insight into other plant signaling pathways, and their evolutionary relationship to well-studied mammalian systems. PUBLIC HEALTH RELEVANCE: Despite their divergence from a common ancestor over 1 billion years ago, both plants and animals utilize steroids as hormones to regulate gene expression that controls growth, development, and homeostasis. The pathways of synthesis and turnover of steroids are remarkably conserved, yet steroid receptors appear to have evolved independently in plants and animals, which may be due to the unique predator/prey relationship of animals and plants. The experiments described in this proposal will thus not only inform our understanding of a new paradigm for steroid hormone perception, but they may also influence our thinking concerning the evolution of signaling pathways in humans as a result of our diet.